Manifestations of chronic rejection (CR) include fibrosis of the graft and vascular neointimal development, referred to as transplant associated vasculopathy (TAV). Despite continued investigation, the underlying mechanisms responsible for these disease manifestations remain poorly defined. TGFbeta has been implicated in fibrosis of the graft, while smooth muscle (SM) cell migration and proliferation are thought to be critical in TAV. Hence, the central hypothesis of this proposal is that selectively targeting the fibrosis inducing, but not the immunosuppressive activities, of TGFbeta and targeting SM cell proliferation will provide an effective treatment to inhibit the progress/on of CR. These studies will be performed in the mouse cardiac allograft model, where transient depletion of recipient CD4+ T cells results in prolonged graft survival and the development of CR. In contrast, when recipients are treated with multiple doses of anti-CD40L allografts remain free of CR unless allografts are transfected with active TGFbeta. Data generated in these models reveal a strong correlation between TGFbeta -induced connective tissue growth factor (CTGF) and CR. Since CTGF is the mediator of TGFbeta -induced fibrosis, targeting CTGF will be explored to test the hypothesis that TGFbeta - induced fibrosis may be segregated from the beneficial anti-inflammatory activities of this cytokine. In addition, gene transfer of the cyclin dependent kinase (cdk) inhibitor p21 will be employed to test the hypothesis that limiting vascular SM cell proliferation will inhibit TAV. Specific Aim 1 will elucidate the role of TGFbeta -induced CTGF in CR and explore the therapeutic potential of targeting CTGF in the progression of CR to test the hypothesis that TGFbeta contributes to the development of CR through the induction of CTGF. Specific Aim 2 will elucidate the cellular requirements for CR and determine the role of Treg in this process to test the hypothesis that while Treg control graft-reactive T cell responses which mediate acute rejection, they produce TGFbeta which contributes to CR. Specific Aim 3 will assess the efficacy of intragraft gene transfer of the cdk inhibitor p21 on TAV to test the hypothesis that p21 will inhibit SM cell proliferation and migration into the neointima of arteries of the graft, thereby limiting the progression of TAV. Relevance to Public Health: Chronic rejection (CR) is the main cause of late transplant failure. Currently, there is no treatment for CR. This study will identify novel therapeutic targets for the treatment of this disease.